IP3-binding polypeptides and methods of using them

ABSTRACT

The present invention provides a high affinity polypeptide having a binding activity to inositol 1,4,5-trisphosphate, to a gene encoding the polypeptide, to a recombinant vector including the gene, to a transformant including the vector and to a method for producing the high affinity polypeptide having a binding activity to inositol 1,4,5-trisphosphate.

This application is a divisional (and claims the benefit of priorityunder 35 USC 120) of U.S. application Ser. No. 09/385,222, filed Aug.26, 1999 (now U.S. Pat. No. 6,465,211), which claims priority to aforeign application filed in Japan, Application No. 242207/1998, filedAug. 27, 1998. The disclosure of the prior application is consideredpart of (and is incorporated by reference in) the disclosure of thisapplication.

FIELD OF THE INVENTION

The present invention relates to a high affinity polypeptide having abinding activity to inositol 1,4,5-trisphosphate, to a gene encoding thepolypeptide, to a recombinant vector including the gene, to atransformant including the vector and to a method for producing the highaffinity polypeptide having a binding activity to inositol1,4,5-trisphosphate.

BACKGROUND OF THE INVENTION

Inositol 1,4,5-trisphosphate (hereinafter, also referred to as “IP₃”) isone of second messengers which are produced by inositol phospholipidmetabolism activated in response to an extracellular stimului such ashormones, growth factors, neurotransmitters or the like. IP₃ is asubstance that induces the increase of intracellular calciumconcentration. The IP₃-induced calcium increase is a crucial and highlyuniversal signal transmission mechanism that is involved in many cellfunctions in a wide variety of animals. For example, IP₃ controls manyphysiological functions such as fertilization, blastogenesis,development and differentiation, cell growth, secretion, immune system,muscle contraction, and cranial nerve functions (gustation, vision,memory, learning, etc.) in diverse organisms, for example, invertebratasuch as nematoda (nemathelminthes), Drosophila (arthropoda) andcuttlefish (mollusca), and vertebrata such as mouse and human.

On the molecular level, this mechanism is initiated by the bindingbetween an IP₃ and its target, an IP₃ receptor. Specifically, when theIP₃ binds to the IP₃ receptor (a calcium channel susceptible to IP₃)present in an intracellular calcium-storing site (endoplasmic reticulum,etc.), the channel opens and releases calcium from the calcium-storingsite into the cytoplasm, thereby controlling the activities ofcalcium-dependent proteins and enzymes.

Heparin, adenophostin (a kind of fungal metabolite) and Xestospongin (akind of sponge metablite) are examples of substances that might affectthe signal transmission by the IP₃-induced calcium. However, althoughheparin inhibits the binding between the IP₃ and the IP₃ receptor, itsspecificity is low since there are various targets in the cell.Adenophostin is an antagonistic agonist of the binding between the IP₃and the IP₃ receptor, and is a powerful activator of the IP₃ receptorchannel. However, its use is limited since its yield from fungus is lowand it cannot transport across the membrane. Xestospongin has recentlybeen reported as an inhibitor of the IP₃ receptor channel that does notinfluence the binding of IP₃. Again, its yield is low and there arestill questions remaining as to its specificity. Thus, currently, thereis almost no substance that is considered to effectively act onIP₃-induced calcium signal transmission. In particular, there has beenno substance or system that inhibits IP₃-induced calcium signaltransmission by specifically trapping IP₃ that has increased on the celllevel.

SUMMARY OF THE INVENTION

The present invention provides a high affinity polypeptide having abinding activity to inositol 1,4,5-trisphosphate, a gene encoding thepolypeptide, a recombinant vector containing the gene, a transformantcontaining the vector and a method for producing the high affinitypolypeptide having a binding activity to inositol 1,4,5-trisphosphate.

In order to solve the above-described problem, the present inventorshave gone through intensive studies and have succeeded in isolating ahigh affinity polypeptide having an extremely high binding activity toIP₃ from a protein including a part of the N-terminal amino acid regionof an IP₃ receptor.

The present invention provides a recombinant polypeptide of thefollowing (a), (b) or (c):

-   (a) a polypeptide comprising an amino acid sequence shown in SEQ ID    NO: 2;-   (b) a polypeptide comprising an amino acid sequence having deletion,    substitution or addition of at least one amino acid in the amino    acid sequence shown in SEQ ID NO: 2, and having a high affinity    binding activity to inositol 1,4,5-trisphosphate; or-   (c) a polypeptide having at least 70% homology with the amino acid    sequence shown in SEQ ID NO: 2, and having a high affinity binding    activity with inositol 1,4,5-trisphosphate.

The present invention also provides a gene coding for a polypeptide ofthe above (a), (b) or (c); or a gene coding for a polypeptide having atleast 70% homology with the gene and having a high affinity bindingactivity with inositol 1,4,5-trisphosphate.

The present invention further provides a gene comprising DNA of thefollowing (d) or (e):

-   (d) DNA of a nucleotide sequence shown in SEQ ID NO: 1; or-   (e) DNA of a nucleotide sequence having at least 70% homology with    the DNA of the nucleotide sequence shown in SEQ ID NO: 1, and coding    for a polypeptide having a high affinity binding activity with    inositol 1,4,5-trisphosphate.

The present invention provides a recombinant vector comprising any oneof the above-described genes.

The present invention also provides a transformant comprising the aboverecombinant vector.

The present invention further provides a method for producing any one ofthe above-mentioned polypeptides, the method comprising: culturing theabove-mentioned transformant; and collecting, from the obtained culture,a polypeptide having a high affinity binding activity to inositol1,4,5-trisphosphate.

This and other advantages of the present invention will become apparentto those skilled in the art upon reading and understanding the followingdetailed description with reference to the accompanying figures.

This specification includes part or all of the contents as disclosed inthe specification and/or drawings of Japanese Patent Application No.10-242207 which is a priority document of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structures of IP₃ sponges;

FIGS. 2A–2C show high expression and IP₃-binding activity of T604;

FIGS. 3A–3C are graphs showing the IP₃-binding activities of the IP₃sponges;

FIG. 4 is a graph showing a curve of IP₃-binding inhibition depending onthe IP₃ sponge concentration;

FIGS. 5A–5F are graphs showing the effects of low-affinity G224-m30 andGST on IP₃-induced Ca²⁺ release;

FIGS. 6A–6G are graphs showing the effect of high affinity IP₃ spongeG224 on inhibition of IP₃-induced Ca²⁺ release; and

FIG. 7 is a plot diagram showing an IP₃-induced Ca²⁺, release dependingon the concentration of the high affinity IP₃ sponge G224.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Hereinafter, the present invention will be described in more detail.

A polypeptide of the present invention specifically binds to IP₃with avery high affinity, and includes a part (a cut) of N-terminal amino acidregion of the natural IP₃ receptor (thus also referred to as a cut-typepolypeptide). The polypeptide of the invention is often referred to as ahigh affinity IP₃-binding polypeptide.

1. Cloning a Gene Coding for the IP₃ Receptor

In order to obtain a high affinity IP₃-binding polypeptide of theinvention, a gene encoding the natural IP₃ receptor protein is cloned.The nucleotide sequence of the IP₃ receptor gene is already known(Nucleic Acid Res. 17: 5385–5386, 1989; Nature 342:32–38, 1989). Thegene may, for example, be prepared according to the following geneengineering procedure.

(i) Preparation and Screening of cDNA Library Encoding the IP₃ Receptor

A known procedure may be employed to prepare mRNA of the IP₃ receptor.For example, total RNA is obtained by treating a tissue or a cell from amouse brain with a guanidine reagent, a phenol reagent or the like.Then, poly(A)+RNA(mRNA) is obtained according to an affinity columnmethod or a batch method using poly (U) sepharose, etc. By using theobtained mRNA as a template as well as oligo dT primer and reversetranscriptase, a single-stranded cDNA is synthesized. Based on thesingle-stranded cDNA, a double-stranded cDNA is synthesized andintroduced into a suitable cloning vector to prepare a recombinantvector to transform E.coli or the like. The transformant is selectedbased on indices such as tetracycline and ampicillin resistance, therebyobtaining a cDNA library.

The transformation of E.coli may be conducted according to the method ofHanahan [Hanahan, D., J. Mol. Biol. 166:557–580 (1983)]. Specifically,the recombinant vector is added to a prepared competent cell under thepresence of calcium chloride, magnesium chloride or rubidium chloride.When a plasmid is used as the vector, it should contain a gene resistantto drugs such as tetracycline and ampicillin. Besides plasmids, acloning vector such as λ phage may also be used.

The thus-obtained transformant is screened for strains with the DNA ofinterest by, for example, “expression cloning” through immunoscreeningusing an antibody, or by polymerase chain reaction (PCR) using a primersynthesized from a known sequence.

The thus-obtained DNA fragment or DNA amplified fragment coding for theantibody epitope is labeled with ³²P, ³⁵S, biotin or the like to be usedas a probe for hybridizing with the transformant DNA denatured and boundon a nitrocellrose filter. Then, the obtained positive strains may bescreened for the target DNA fragment.

(ii) Determination of the Nucleotide Sequence

The obtained clone is determined for its nucleotide sequence. Thenucleotide sequence may be determined according to a known method suchas Maxam-Gilbert chemical modification method, dideoxynucleotide chaintermination method using M13 phage. Generally, the sequence isdetermined using an automatic DNA sequencer (e.g., Perkin-Elmer 373A DNAsequencer).

The nucleotide sequence of the natural (full-length) gene coding for theIP₃ receptor and the full-length amino acid sequence of the IP₃ receptorare shown in SEQ ID NOS. 3 and 4, respectively.

2. Design and Synthesis of a Gene Coding for a High Affinity IP₃-BindingPolypeptide of the Invention

(i) Design and Synthesis of a Gene Coding for a High AffinityIP₃-binding Polypeptide

A high affinity IP₃-binding polypeptide of the invention includes a cutof N-terminal amino acid region, that is, Amino acids 579 to at least800, preferably Amino acids 579 to at least 734, of the amino acidsequence of the full-length IP₃ receptor protein (SEQ ID NO:4).According to the present invention, this cut-type polypeptide is alsoreferred to as an IP₃ sponge (FIG. 1). Due to this cut, the polypeptide(IP₃ sponge) of the invention gains a very strong specific bindingability to IP₃ (high affinity IP₃-binding activity).

Herein, the phrase “high affinity” is used in the situation where theIP₃ sponge has an IP₃ affinity that is about 100 to 1,000 times(preferably 500 to 1,000 times) higher than that of the natural IP₃receptor.

According to the present invention, the IP₃ sponge also includes atleast the amino acid sequence shown in SEQ ID NO: 14, which correspondsto Amino acids 226–578 of the amino acid sequence of SEQ ID NO: 4.Herein, this region is referred to as a “core” region.

Based on the above-described facts, the length of the fragment of theinvention and the length of the DNA coding for the fragment can bedetermined at one's discretion providing that the high affinityIP₃-binding activity is maintained. The fragment may include, forexample, Amino acids 224–604 of the amino acid sequence of SEQ ID NO: 4(encoded by Nucleotides 998–2140 of the nucleotide sequence of SEQ IDNO: 3); Amino acids 1–604 of the amino acid sequence of SEQ ID NO: 4(encoded by Nucleotides 329–2140 of the nucleotide sequence of SEQ IDNO: 3); or Amino acids 1–734 of the amino acid sequence of SEQ ID NO: 4(encoded by Nucleotides 329–2540 of the nucleotide sequence of SEQ lIDNO: 3).

These fragments are obtained through PCR using primers that are designedbased on nucleotide regions of the nucleotides shown in SEQ ID NO. 3outside the regions of the respective fragments, as well as the DNAcoding for the natural IP₃ receptor (SEQ ID NO: 3, Nucleic Acid Res. 17:5385–5386, 1989; Nature 342: 32–38, 1989) as a template.

(ii) Preparation of a Gene Encoding a Mutant-type IP₃ Sponge of theInvention (Mutant-type IP₃ Gene)

According to the present invention, the amino acid sequence of the IP₃sponge may, at least partially, be introduced with a mutation. Such amutant-type IP₃ sponge is also contemplated as the IP₃ sponge of thepresent invention. A mutation is introduced into the amino acidsequence, by mutating the nucleotide sequence of the gene coding for theamino acid sequence of the IP₃ sponge.

The mutation is introduced into the gene according to a known methodsuch as Kunkel method, Gapped duplex method or any method equivalentthereof. For example, site-directed mutatagenesis may be employed inwhich a mutant oligonucleotide is used as a primer (Yoshikawa, F. etal., J. Biol. Chem. 271: 18277–18284, 1996). Alternatively, a mutationmay be introduced by using a mutagenesis kit such as hMutant-K (Takara),Mutant-G (Takara) and a series of LA PCR in vitro Mutagenesis kits(Takara).

First, based on the nucleotides of the gene coding for the IP₃ sponge ofthe invention (also referred to as an “IP₃ sponge gene”), a primer issynthesized such that the primer includes a mutated nucleotide or siteand about 10 nucleotides flanking the mutated nucleotide or site. Usingthis primer as well as the IP₃ sponge gene as a template, PCR reactionis conducted. The resultant is purified and then treated with a suitablerestriction enzyme, thereby obtaining the mutant-type IP₃ sponge gene ofinterest.

(iii) Determination of the Nucleotide Sequences

The nucleotide sequence of the genes obtained through (i) and (ii) isdetermined. The determination is conducted by a known method such asMaxam-Gilbert chemical modification method, dideoxynucleotide chaintermination method using M13 phage, or any other method. Generally, anautomatic sequencer (e.g., 373A DNA sequencer produced by Perkin-Elmer)is used.

A nucleotide sequence of an IP₃ sponge gene of the invention and anamino acid sequence of the IP₃ sponge of the invention are shown in SEQID NOS: 1 and 2, respectively. The polypeptide of this amino acidsequence may include at least one deletion, substitution, addition orthe like as long as it has a high affinity with IP₃ and has an activityof specifically binding to IP₃.

For example, at least one, preferably about 1 to 10, more preferably 1to 5 of the amino acids in the core region (the amino acid sequenceshown in SEQ lID NO: 14) may be deleted; at least one, preferably about1 to 10, more preferably 1 to 5 amino acids may be added to the aminoacid sequence of the core region; or at least one, preferably 1 to 10,more preferably 1 to 5 of the amino acids in the core region may bereplaced with other amino acids.

The polypeptide of the present invention is not limited by the length ofthe amino acid sequence as long as the amino acid sequence contains theamino acid sequence of the core region and a cut of N-terminal Aminoacids 579 to at least 800, preferably N-terminal Amino acids 579 to atleast 734 of the natural-type IP₃ receptor (SEQ ID NO:4). For example,Amino acids 224–604 (polypeptide “G224”) of the amino acid sequenceshown in SEQ ID NO:4 and the gene encoding G224 are also contemplated asthe lIP3 sponge and the 1P3 sponge gene of the invention, respectively.

The polyp eptide G224 may have a mutation of at least one, preferablyabout 1 to 10, more preferably 1 to 5 amino acids. Thus, the IP₃ spongeof the invention may inclulde an amino acid sequence where lysine atPosition 508 of the amino acid sequence G224 is replaced with alanine(mutation “m30”) or where arginine at Position 441 of the amino acidsequence G224 is replaced with glutamine (mutation “m49”) (FIG. 1).Herein, the numbers indicating the positions of the amino acids arebased on the amino acid sequence shown in SEQ ID NO:4 (e.g., Position 1is the first amino acid of SEQ ID NO: 4).

A polypeptide including an amino acid sequence having 70% or morehomology with the core region (SEQ ID NO: 2), and having a high affinitybinding activity with inositol 1,4,5-trisphosphate is also contemplatedas the present invention.

Also contemplated as the present invention is a gene coding for thepolypeptide having the above-described mutation in its amino acidsequence, and having a high affinity binding activity with IP₃ receptor.In addition, a nucleotide sequence coding for the amino acids includedin the IP₃ sponge of the present invention, and a degenerate isomercoding for the same polypeptide with different degenerate codons arealso contemplated as the genes of the invention. Also contemplated asthe present invention is DNA having at least 70% homology with thenucleotide sequence of these genes, for example, DNA of other typebelonging to the IP₃ receptor gene family that codes for a regioncorresponding to the polypeptide of the present invention.

Once the nucleotide sequence of the gene of the present invention isdetermined, the gene may be obtained by PCR using a primer that issynthesized chemically or that is synthesized from the determinednucleotide sequence.

3. Preparation of Recombinant Vector and Transformant Containing IP₃Sponge Gene of the Invention

(i) Preparation of Recombinant Vector

A recombinant vector of the invention may be obtained by ligating(inserting) the IP₃ sponge gene of the invention to (into) a suitablevector. The vector for inserting the gene of the invention is notlimited to a specific one as long as it is replicable in a host cell.Examples of such vector include but not limited to plasmid DNA and phageDNA.

The plasmid DNA is, for example, plasmid from E.coli (e.g., pET-3a,pBR322, pBR325, pUC118, pUC119, etc.), plasmid from bacillus (e.g.,pUB110, pTP5, etc.), or plasmid from yeast (e.g., YEp13, YEp24, YCp50,etc.). The phage DNA is, for example, λ phage. Similarly, an animalvirus vector such as retrovirus, adenovirus or vaccinia virus vectors,or an insect virus vector such as a baculovirus vector may also be used.A fusion plasmid in which GST, GFP, His-tag, Myc-tag or the like islinked with each other may also be used (e.g., pGEX-2T, pEGFP-N3).

To insert the gene of the invention into the vector, first, the purifiedDNA is cleaved with suitable restriction enzymes. Then, the cleavedfragment is inserted into a restriction site or a multicloning site ofthe suitable vector DNA.

The gene of the present invention should be integrated into the vectorsuch that the gene can function. If desired, the vector of the inventionmay include, other than the gene of the invention and the promoter, forexample, a cis-element (e.g., an enhancer), a splicing signal, a poly(A)tail signal, a selective marker, and a ribosome binding sequence (SDsequence). Examples of the selective marker include a dihydrofolatereductase gene, an ampicillin-resistant gene and a neomycin-resistantgene.

(ii) Preparation of Transformant

A transformant of the invention may be obtained by introducing therecombinant vector of the invention into a host cell in such a mannerthat the gene of interest is capable to be expressed. The host cell isnot limited to a specific one as long as it can express the gene of thepresent invention. Bacteria such as genus Escherichia (e.g., Escherichiacoli), genus Bacillus (e.g., Bacillus subtilis), genus Pseudomonas(e.g,. Pseudomonas putida), yeast such as Saccharomyces cerevisiae andSchizosaccharomyces pombe, animal cells (e.g., COS, CHO, HEK293, PC12cells), and insect cells (e.g., Sf9 and Sf21) are exemplified.

When a bacterium such as E.coli is used as the host, it is preferablethat the recombinant vector of the present invention is capable ofautonomous replication in the host and that it includes a promoter, aribosome binding sequence, the gene of the invention and a transcriptiontermination sequence. The recombinant vector may also include a gene forcontrolling the promoter.

As the E.coli, E.coli BL21, JM109 and HB101 are exemplified and asbacillus, Bacillus subtilis MI 114 and 207-21 are exemplified.

Any promoter may be used as long as it can be expressed in a host celllike E.coli. For example, a promoter derived from E.coli or phage, e.g.,trp promoter, lac promoter, p_(L) promoter or p_(R) promoter, may beused. Artificially designed and modified promoter like tac promoter mayalso be used.

The recombinant vector may be introduced into the host bacteriumaccording to any method for introducing DNA into a bacterium. Forexample, calcium ion method (Cohen, S. N. et al., Proc. Natl. Acad.Sci., USA, 69: 2110–2114 (1972)) and an electroporation method may beemployed.

An yeast such as Saccharomyces cerevisiae, Schizosaccharomyces pombe orPichia pastoris may also be used as the host. In this case, the promotermay be any promoter that can be expressed in the yeast. Examples of suchpromoter include but not limited to gall promoter, ga110 promoter, heatshock protein promoter, MF 1 promoter, PHO5 promoter, PGK promoter, GAPpromoter, ADH promoter and AOX1 promoter.

The recombinant vector may be introduced into the yeast by any methodfor introducing DNA into an yeast. For example, electroporation method(Becker, D. M. et al., Methods Enzymol., 194, 182–187 (1990)),spheroplast method (Hinnen, A. et al., Proc. Natl. Acad. Sci., USA, 75,1929–1933 (1978)), or lithium acetate method (Itoh, H., J. Bacteriol.,153, 163–168 (1983)) may be employed.

An animal cell such as simian cell (e.g., COS-7, Vero), Chinese hamsterovary cell (CHO cell), mouse L cell, rat cell (e.g., GH3, PC12 orNG108-15) or human cell (e.g., FL, HEK293, HeLa or Jurkat) may also beused as the host. As a promoter, for example, SR promoter, SV40promoter, LTR promoter or β-actin promoter may be used. Other than thesepromoters, an early gene promoter of human cytomegalovirus may also beused.

The recombinant vector may be introduced into the animal cell, forexample, by an electroporation method, a calcium phosphate method or alipofection method.

An insect cell such as Sf9 cell, Sf21 cell or the like may also be usedas the host. The recombinant vector may be introduced into the insectcell, for example, by a calcium phosphate method, a lipofection methodor an electroporation method.

4. Production of IP₃ Sponge

The IP₃ sponge of the present invention may be obtained by culturing theabove-described transformant, and recovering the IP₃ sponge from theculture product. The term “culture” as used herein refers to a culturesupernatant, a cultured cell or microbial cell, or a cell or microbialcell debris.

The transformant of the invention is cultured according to a generalmethod employed for culturing the host.

A medium for culturing the transformant obtained from a microorganismhost such as E.coli or yeast may be either a natural or a syntheticmedium providing that it contains carbon sources, nitrogen sources,inorganic salts and the like assimilable by the microorganism, and thatit can efficiently culture the transformant.

As carbon sources, carbohydrate such as glucose, fructose, sucrose,starch; organic acids such as acetic acid, propionic acid; and alcoholssuch as ethanol and propanol may be used.

As nitrogen sources, ammonia; ammonium salts of inorganic or organicacids such as ammonium chloride, ammonium sulfate, ammonium acetate andammonium phosphate; other nitrogen-containing compounds; Peptone; meatextract; corn steep liquor and the like may be used.

As inorganic substances, potassium dihydrogen phosphate, dipotassiumhydrogen phosphate, magnesium phosphate, magnesium sulfate, sodiumchloride, iron(II) sulfate, manganese sulfate, copper sulfate, calciumcarbonate and the like may be used.

The cultivation is generally performed under aerobic conditions such asshaking or aeration agitating conditions at 37° C. for 6 to 24 hours.During the cultivation, pH is maintained at 7.0 to 7.5. pH is regulatedwith an inorganic or organic acid, an alkali solution or the like. Ifnecessary, an antibiotic such as ampicillin, tetracycline or the likemay be added to the medium during the cultivation.

When culturing a microorganism transformed with an expression vectorusing an inducible promoter, an inducer may be added to the medium atneed. For example, isopropyl 1-thio-β-D-galactoside (IPTG) may be addedto the medium when culturing a microorganism transformed with anexpression vector pET-3a having T7 promoter (that is inducible withIPTG). When culturing a microorganism transformed with an expressionvector using trp promoter (that is inducible with indole acetic acid(IAA)), IAA may be added to the medium.

A transformant obtained with an animal cell host may be cultured in agenerally used medium such as RPMII640 medium or DMEM medium, or amedium obtained by supplementing the generally used medium with fetalbovine serum and the like.

The cultivation is generally conducted under 5% CO₂ at 37° C. for 1 to30 days. If necessary, an antibiotic such as kanamycin, penicillin orthe like may be added to the medium during the cultivation.

After the cultivation, in the case where a microbial cell or a cellintracelluraly produced the IP₃ sponge of the invention, the IP₃ spongeis collected by disrupting the microbial cell or the cell by sonication,freezing and thawing method, or homogenizing. In the case where amicrobial cell or a cell extracellularly produced the IP₃ sponge of theinvention, the microbial cell or the cell is removed from the culturethrough centrifugation or the like before, or the culture solution isdirectly subjected to the isolation/purification procedure. The IP₃sponge of the invention is isolated and purified from the culturethrough a general biochemical method for isolating and purifying aprotein, such as ammonium sulfate precipitation, gel chromatography, ionexchange chromatography, affinity chromatography, or a combinationthereof.

5. Therapeutic Agent and Agent for Gene-Therapy

Since the protein and the gene of the invention has IP₃ neutralizingactivity, they are useful as an antagonist for IP₃-induced calcium, atherapeutic agent and an agent for gene therapy for diseases associatedwith calcium production. The therapeutic agent or the agent for genetherapy of the invention can be administered orally or parenterally andsystemically or locally.

When the protein or the gene of the invention is used as a therapeuticagent or an agent for gene therapy for disease associated with calciumproduction, the disease to be treated is not particularly limited. Forexample, the protein or the gene may be used for diseases in the nervoussystem, blood vascular system, respiratory system, digestive system,lymphatic system, urinary system, reproduction system or the like forthe specific purpose of treatment or prevention. These diseases may bein the form of a single disease or may be complicated by one of thesediseases or by some disease other than those mentioned above; any ofsuch forms may be treated with the protein or the gene of the invention.

When the therapeutic agent of the invention is administered orally, theagent may be formulated into a tablet, capsule, granule, powder, pill,troche, internal liquid agent, suspension, emulsion, syrup or the like.Alternatively, the therapeutic agent may be prepared into a dry productwhich is re-dissolved just before use. When the therapeutic agent of theinvention is administered parenterally, the agent may be formulated intoa intravenous injection (including drops), intramuscular injection,intraperitoneal injection, subcutaneous injection, suppository, or thelike. Injections are supplied in the form of unit dosage ampules ormulti-dosage containers.

These formulations may be prepared by conventional methods usingappropriate excipients, fillers, binders, wetting agents, disintegratingagents, lubricating agents, surfactants, dispersants, buffers,preservatives, dissolution aids, antiseptics, flavoring/perfumingagents, analgesics, stabilizers, isotonicity inducing agents, etc.conventionally used in pharmaceutical preparations.

Each of the above-described formulations may contain pharmaceuticallyacceptable carriers or additives. Specific examples of such carriers oradditives include water, pharmaceutically acceptable organic solvents,collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinylpolymers, sodium alginate, water-soluble dextran, sodium carboxymethylamylose, pectin, xanthan gum, gum arabic, casein, gelatin, agar,glycerol, propylene glycol, polyethylene glycol, vaseline, paraffin,stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitoland lactose. One or a plurality of these additives are selected orcombined appropriately depending of the form of the preparation.

The dosage levels of the therapeutic agent of the invention will varydepending on the age of the subject, the route of administration and thenumber of times of administration and may be varied in a wide range.When an effective amount of the protein of the invention is administeredin combination with an appropriate diluent and a pharmaceuticallyacceptable carrier, the effective amount of the protein can be in therange from 0.0001 to 1000 mg/kg per administration. The therapeuticagent is administered once a day or in several dosages per day for atleast one day.

When the gene of the invention is used as an agent for gene therapy fordiseases associated with calcium production, the gene of the inventionmay be directly administered by injection. Alternatively, a vectorincorporating the gene of the invention may be administered. Specificexamples of a suitable vector for this purpose include an adenovirusvector, adeno-associated virus vector, herpes virus vector, vacciniavirus vector and retrovirus vector. The gene of the invention can beadministered efficiently by using such a virus vector. Alternatively,the gene of the invention may be enclosed in phospholipid vesicles suchas liposomes, and the resultant liposomes may be administered to thesubject. Briefly, since liposomes are biodegradable material-containingclosed vesicles, the gene of the invention is retained in the internalaqueous layer and the lipid bilayer of liposomes by mixing the gene withthe liposomes (a liposome-gene complex). Subsequently, when this complexis cultured with cells, the gene in the complex is taken into the cells(lipofection). Then, the resultant cells may be administered by themethods described below.

As a method for administering the agent for gene therapy of theinvention, local administration to tissues of the central nervous system(brain, spiral cord), blood vascular system (artery, vein, heart),respiratory system (trachea, lung), digestive system (salivary glands,stomach, intestines, liver, pancreas), lymphatic system (lymph node,spleen, thymus), urinary system (kidney), reproduction system (testis,ovary, uterus) or the like may be performed in addition to conventionalsystemic administration such as intravenous or intra-arterialadministration. Further, an administration method combined with cathetertechniques and surgical operations may also be employed.

The dosage levels of the agent for gene therapy of the invention varydepending on the age, sex and conditions of the subject, the route ofadministration, the number of times of administration, and the type ofthe formulation. Usually, it is appropriate to administer the gene ofthe invention in an amount of 0.01–100 mg/adult body/day.

EXAMPLES

Hereinafter, the present invention will be described in detail by way ofexamples which do not limit the technical scope of the presentinvention.

Example 1 Constuction of Expression Plasmid for High AffinityIP₃-Binding Polypeptide (IP₃ Sponge)

The N-terminal amino acids (734 amino acids) (polypeptide T734) of amouse Type-1 IP₃ receptor (mIP₃R1) has a specific IP₃-binding activity.The cDNA portion coding for polypeptide T734 was cloned into E.coliexpression vector pET-3a (whose expression is controlled by T7 promoterthat is induced upon addition of IPTG) to obtain plasmid pET-T734(Yoshikawa F. et al., J. Biol. Chem. 271:18277–18284, 1996). Using thisplasmid (pET-T734) as a parent plasmid, the following expressionplasmids were constructed for IP₃-binding polypeptides. Herein, anIP₃-binding polypeptide with high affinity is also referred to as an“IP₃ sponge”.

(1-1) Expression Plasmid for High Affinity IP₃ Sponge “T604”

A gene coding for polypeptide T604 that corresponds to the firstmethionine (M-1) to the lysine at Position 604 (K-604) of polypeptideT734 was prepared. Specifically, site-directed mutagenesis was conductedby PCR using a complementary oligonucleotide (Yoshikawa F. et al., JBiol Chem, 271:18277–18284, 1996) to introduce a stop codon (TAA) and asubsequent BamHI recognition site (GGATCC) at Position 605 of T734.

Sense primer: 5′-TGTCAGACAT ATGCGTGTTGGAA-3′ (SEQ ID NO: 5)          NdeI Antisense primer: 5′-CGCGGGATCCTTATTTCCGGTTGTTGTGGAGCAGGG-3′ (SEQ ID NO: 6)        BamHI

The sense primer was introduced with a NdeI cleavage recognitionsequence (CATATG) (underlined) including the first methionine codon(ATG).

A total of 100 μl PCR reaction solution was used. The PCR reactionsolution contained 100 ng template DNA, 10 mM KCl, 6 mM (NH₄)₂SO₄, 20 mMTris-HCl (pH 8.2), 2 mM MgCl₂, 0.1% TritonX-100, 10 μg/ml BSA, 200 μMdNTPs, 1 μM sense primer, 1 μM anti-sense primer and 2.5 unit Pfu DNApolymerase. The PCR reaction was performed at 95° C. for 1 min. and thenthrough 30 cycles of: 95° C. for 1 min.; 57° C. for 3 min.; and 72° C.for 3 min.

The 5′-end of the obtained amplified fragment was treated with NdeI andthe 3′-end with BamHI, thereby producing deletion mutant pET-T604 thatcontains DNA coding for an amino acid sequence corresponding to theamino acid sequence of T734 but with C-terminal deletion up to Position605.

(1-2) Expression Plasmid for High Affinity IP₃ Sponge “G224”

First, a gene coding for polypeptide T604 that corresponds to the firstmethionine (M-1) to the lysine at Position 604 (K-604) of polypeptideT734 was prepared. Specifically, site-directed mutagenesis was conductedby PCR using a complementary oligonucleotide (Yoshikawa F. et al., JBiol Chem, 271:18277–18284, 1996) to introduce a stop codon (TAA) and asubsequent EcORI recognition site (GAATCC) at Position 605 of T734.

Sense primer: 5′-TGTCAGACAT ATGCGTGTTGGAA-3′ (SEQ ID NO: 5)          NdeI Antisense primer: 5′-CCGGAATTCTTATTTCCGGTTGTTGTGGAGCAGGG-3′ (SEQ ID NO: 7)       EcoRI

The PCR was conducted under the same conditions as described in (1-1)above.

The 5′-end of the thus-obtained amplified fragment was treated with NdeIand the 3′-end with EcoRI, thereby producing deletion mutant pET-T604ethat contains DNA coding for an amino acid sequence corresponding to theamino acid sequence of T734 but with C-terminal deletion up to Position605.

Then, using deletion mutant pET-T604e as a template, site-directedmutagenesis was performed to introduce BamHI recognition site (GGATCC)immediately before the methionine at Position 224 of polypeptide T604.

Antisense primer: 5′-CCGGAATTC TTATTTCCGGTTGTTGTGGAGCAGGG-3′ (SEQ ID NO:7)       EcoRI Sense primer: 5′-CGCGGATCCATGAAATGGAGTGATAACAAAGACGACA-3′ (SEQ ID NO: 8)       BamHI

The PCR was conducted under the same conditions as described in (1-1)above.

The thus-obtained amplified fragment (plasmid introduced with mutation)was cleaved with BamHI and EcoRI, thereby obtaining a CDNA fragmentcoding for Amino acids 224–604. This cDNA fragment was ligated toBamHI-EcoRI site of GST fusion plasmid (pGEX-2T) without a frameshift(in-frame), thereby obtaining plasmid pGEX-G224. Plasmid pGEX-G224expresses fusion polypeptide G224 (FIG. 1) that includes polypeptide GSTand subsequent polypeptide M-224 to K-604.

(1-3) Expression Plasmid for Low Affinity IP₃-binding Polypeptide

Site-directed mutagenesis was conducted by sequential PCR usingpGEX-G224 as a template.

The following two mismatched oligonucleotides were synthesized tointroduce mutation (K508A) at Position 508 of T604 where alanine wassubstituted for lysine (K-508):

5′-GAGAGCGGCAGGCACTGATGAGGG-3′ (SEQ ID NO:9)5′-CCCTCATCAGTGCCTGCCGCTCTC-3′ (SEQ ID NO:10)

Using the above primers, site-directed mutagenesis was conducted bysequential PCR. The PCR conditions and the composition of the reactionsolution were as follows:

Primary reaction 1 Sense primer: 5′-GAGAGCGGCAGGCACTGATGAGGG-3′ (SEQ IDNO: 9) Antisense primer: 5′-CCGGAATTC TTATTTCCGGTTGTTGTGGAGCAGGG-3′ (SEQID NO: 7)       EcoRI

The PCR was conducted under the same conditions as described in (1-1)above.

Primary Reaction 2

Sense primer: 5′-CGCGGATCC ATGAAATGGAGTGATAACAAAGACGACA-3′ (SEQ ID NO:8)       BamHI Antisense primer: 5′-CCCTCATCAGTGCCTGCCGCTCTC-3′ (SEQ IDNO: 10)

The PCR was conducted under the same conditions as described in (1-1)above.

Secondary Reaction

Ten μl of the PCR reaction product resulting through Primary reactions 1and 2, and 1 μM each of primers (SEQ ID NOS: 7 and 8) were used toconduct PCR under the same conditions as the primary reactions.

The obtained amplified fragment was cleaved with BamHI and EcoRI. Thecleaved fragment was ligated to BamHI-EcoRI site of GST fusion plasmidpGEX-2T without a frameshift (in frame), thereby obtaining plasmidpGEX-G224-m30. This mutant plasmid expresses polypeptide G224-m30 havingthe point mutation K508A (FIG. 1, m30).

(1-4) Expression Plasmid for High Affinity IP₃ Sponge “G224-m49”

Using pGEX-G224 as a template, site-directed mutagenesis was conductedby sequential PCR.

The following two mismatched oligonucleotides were synthesized tointroduce a mutation (R441Q) at Position 441 of T604 where glutamine wassubstituted for arginine (R-441).

5′-GCTGAGGTTCAAGACCTGGACTTTG-3′ (SEQ ID NO: 11)5′-AAAGTCCAGGTCTTGAACCTCAGC-3′ (SEQ ID NO: 12)

Primary Reaction 1

Sense primer: 5′-GCTGAGGTTCAAGACCTGGACTTTG-3′ (SEQ ID NO: 11) Antisenseprimer: 5′-CCGGAATTC TTATTTCCGGTTGTTGTGGAGCAGGG-3′ (SEQ ID NO: 7)      EcoRI

The PCR was conducted under the same conditions as described in (1-3)above.

Primary Reaction 2

Sense primer: 5′-CGCGGATCC ATGAAATGGAGTGATAACAAAGACGACA-3′ (SEQ ID NO:8)       BamHI Antisense primer: 5′-AAAGTCCAGGTCTTGAACCTCAGC-3′ (SEQ IDNO: 12)

The PCR was conducted under the same conditions as described in (1–3)above.

Secondary Reaction

Ten μl of the PCR reaction product resulting through Primary reactions 1and 2, and 1 μM each of primers (SEQ ID NOS: 6 and 8) were used toconduct PCR under the same conditions as those of the primary reactions.

The obtained amplified fragment was cleaved at a BamHI-EcoRI site. Thecleaved fragment was ligated to BamHI-EcoRI site of GST fusion plasmidpGEX-2T without a frameshift (in frame), thereby obtaining plasmidpGEX-G224-m49. This mutant plasmid expresses polypeptide G224-m49 havingthe point mutation R441Q (FIG. 1, m49).

Example 2 Expression and Preparation of High Affinity IP₃-BindingPolypeptide with E.coli

Since the IP₃-binding core mostly results in insoluble inclusion bodies,the expression amount is low. Thus, the present inventors have modifiedthe IP₃-binding region through gene engineering to produce a highaffinity IP₃-binding polypeptide which is of lower molecule, which iscapable of stable mass-expression, which can be recovered as a solubleprotein, which has a higher affinity, and which has as high specificityas a conventional IP₃ receptor.

By low-temperature cultivation (16–22° C.), polypeptide T734 can bemass-expressed in a stable manner with a relatively high solublefraction recovery (Kd=50±2.4 nM, B_(max)=46 pmol/mg protein, 1.85 mg/lE.coli culture (corresponding to about 0.5 g of wet E.coli)). However,the inclusion bodies amount to more than ten times the amount of thesoluble fraction (Yoshikawa F. et al., J. Biol Chem. 271: 18277–18284,1996).

First of all, smaller polypeptides that had the above-describedcharacteristics were prepared.

The pET-type and pGEX-type expression plasmids obtained in Example 1were introduced into E.coli BL21 (DE3) and JM109, respectively, bytransformation method. Expression induction with IPTG and preparation ofexpression proteins from E.coli were mainly conducted by modifying themethod of Yoshikawa et al (Yoshikawa F. et al., J. Biol Chem. 271:18277–18284, 1996).

Specifically, E.coli introduced with respective plasmids were shakecultured in L broths (containing 100 μg/ml ampicillin) at 22° C. Whenthe absorption OD₆₀₀ became about 1.5, IPTG was added to 0.5 mM. After afew hours of shake culture at 16° C., each of the E.coli was recoveredthrough centrifugation and suspended in PBS containing proteaseinhibitors. (1 mM PMSF, 10 μM leupeptin, 1 μM pepstatin A, 2 μg/mlaprotinin). Each of the E.coli was disrupted by sonication. Then, eachsupernatant containing the expression polypeptide (soluble fraction) wascollected by ultracentrifugation (Beckman Ti35 rotor, 25,000 rpm, 1 hr.,4° C.).

GST fusion polypeptides were purified from the soluble fractions byaffinity purification using Glutathione-Sepharose column (PharmaciaLKB). Specifically, each of the GST fusion polypeptides was eluted fromthe column with 10 mM glutathione/50 mM Tris-HCl (pH 8.0) by mainlyfollowing the manual provided by the manufacturer. The polypeptidesolutions were equilibrated with 10 mM HEPES-KOH (pH 7.2), 88 mM NaCland 1 mM KCl using PD10 desalted column (Pharmacia LKB), and thendispensed, thereby obtaining the IP₃ sponges (FIG. 1: G224, m30, m49 andGST). The IP₃ sponges were stored at −80° C. until they were used.

A series of deletion mutants based on polypeptide T734 were prepared byserially shortening the length of the polypeptide T734 from theC-terminus. The analysis of the deletion mutants indicated that T705 andT699 had no marked characteristic difference with T734. In the cases ofpolypeptides T569, T572 and T576, the expression amounts of the solubleproteins were lower than T734. Stable mass-expression of soluble proteinwas successful with polypeptide T604 which was obtained by deleting theC-terminus of T734 up to Amino acid 605 (FIG. 1).

With reference to FIG. 1, the uppermost (IP₃R1) is the N-terminal aminoacids of the IP₃ receptor including the IP₃-binding core region (core:Amino acids 226–578). T604 (Amino acids 1–604), G224 (GST+Amino acids224–604), G224m30 (G224 introduced with K508A mutation), G224m49 (G224introduced with R441Q mutation), and GST (derived from pGEX-2T)) arealso shown in FIG. 1.

T604 had a [³H]IP₃-binding activity substantially equivalent to that ofT734 (Kd=45 nM), and a higher yield of soluble protein (B_(max)=690pmol/mg protein). Specifically, the yield was 19 mg/l E.coli culture(FIGS. 2B and 2C, Table 1).

TABLE 1 Expression of IP₃-binding site in E. coli Expression B_(max)[pmol/μg efficiency (mg/l Kd purified Protein E. coli culture) [nM]protein] Purified IP₃R^(a) — 83 2.1 T734^(b) 1.85 (50)^(c) ND T604 197.6/(45)^(c) ND G224 30 0.083 1.6 G224m49 ND 0.043 1.7 G224m30 ND 3303.0 ^(a)Maeda et al., EMBO J. 9, 51–67, 1990 ^(b)Yoshikawa et al., J.Biol. Chem. , 271, 18277–18284, 1996 ^(c)the values in parenthesesrepresent Kd obtained from crude cell lysates ND. Not Determined

The total expression amount of polypeptide T604 substantially equaled tothat of polypeptide T734 but T604 had a remarkably improved solubleprotein yield. The yield of soluble protein of polypeptide T604 wassubstantially the same at 30° C. and 37° C., and reached the peak within2 hours after initiating expression induction.

FIG. 2A shows the result of Western blotting analysis of the protein(0.1 μg) obtained from an E.coli extract solution (soluble fraction)that expresses polypeptide T604 (66 kDa). As a control, a cell extractsolution obtained by transforming a vector that does not include T604(pET-3a) was used. FIG. 2B shows a comparison of the total amounts ofspecific IP₃-binding contained in 0.7 μl soluble fractions, for T734,T604, and the control vector. FIG. 2C shows the result of Schatchardplot analysis where the binding between 3 μg of T604 soluble fractionand 9.6 nM [³H]IP₃ was competitively inhibited with non-labeled IP₃(cold IP₃) at various concentrations. The results were Kd=45±7.6,B_(max)=690±64 pmol/mg protein.

When T734 was serially deleted from the N-terminus, a very shortN-terminal deletion of T734 (e.g., a deletion of 31 amino acids) causedlack of IP₃-binding activity even the deletion was outside the coreregion. However, the polypeptide retrieved the IP₃-binding activity whenthe N-terminus was deleted to Amino acid 220–225, near the N-terminus ofthe core region (Yoshikawa et al, 1996). The theory for this is unknown,but presumably, the formation of the three-dimensional structure foractive core region is somehow interrupted depending on the degree ofdeletion. Although the active polypeptide with the N-terminal deletionup to Amino acid 220–225 had a relatively high affinity, the amount ofsoluble protein expressed was lower.

As described above, a protein obtained by deleting Amino acids 1–223 ofpolypeptide T604 (N4-T604; Amino acids 224–604) had a higher activity(about 3 times high) but lower production than those of the originalT604. Accordingly, polypeptide T604 seemed to be the most suitablepolypeptide for stably mass-expressing only the high affinityIP₃-binding region as a soluble protein.

Example 3 Expression of IP₃ Sponge

(i) [³H]-IP₃-binding Inhibition Experiment

Based on the results obtained in Example 2, an IP₃-binding polypeptidewith a higher affinity was produced. As described above, when theamino-terminal Amino acids 1–223 of polypeptides T604 and T734 weredeleted, high [³H]IP₃-binding activities were obtained. Even Amino acidregion 224–579 (a polypeptide that almost corresponds to the coreregion) consisting of only 356 amino acid residues has an affinity ashigh as Kd=2.3 nM (Yoshikawa et al., 1996, supra). However, as describedabove, these polypeptides have lower soluble protein expression levels.In other words, longer amino terminal deletion may result in a higheraffinity on one hand, but it also lowers the expression amount andexpression stability of soluble proteins by rendering most of proteinsas insoluble inclusion bodies.

In general, stability, solubility and an expression level of a foreignpolypeptide are known to be improved when it is made into a GST fusionbody. In this example, fusion proteins G224, G224-m30 and G224-m49consisting of GST and an IP₃-binding site (Amino acid region 224–604)were prepared by ligating GST to replace the N-terminal region (Aminoacids 1–223) of the IP₃ receptor (FIG. 1).

The IP₃-binding activities of these fusion proteins were measured mainlyby the method of Yoshikawa et al (1996).

Each fusion protein (IP₃ sponge) (0.2 μg) was mixed with 100 μl ofbinding buffer-α (50 mM Tris-HCl (pH 8.0 at 4° C.), 1 mM EDTA, 1 mMβ-mercaptoethanol) that contained 9.6 nM D-myo-[³H](1,4,5)IP₃ (777GBq/mmol; DuPont NEN) (hereinafter, abbreviated as “[³H]IP₃”) andvarious concentrations of non-labeled D-myo-(1,4,5)IP₃ (Dojindo)(hereinafter, abbreviated as “cold IP₃”). The mixture was left to standon ice for 10 minutes. To the mixture, 4 μl of 50 mg/ml γ-globulin(Sigma) (final concentration: 1 mg/ml) and 100 μl of 30% PEG 6000(Sigma)/binding buffer-a solution (final concentration: 15%) were added.The resultant mixture was left to stand on ice for 5 minutes, and thencentrifuged at 10,000×g at 2° C. for 5 minutes to collectpolypeptide/PEG complex. PEG-precipitated [³H]IP₃-binding polypeptidewas well solubilized with 180 μl solubilizer Solvable (DuPont NEN). Theresultant was neutralized with 18 μl glacial acetic acid and then addedto 5 ml liquid scintillation counter (Atomlight [DuPont NEN]) to measurethe radioactivity (first radioactivity). Non-specific binding of eachprotein was determined by measuring the second radioactivity in thepresence of 2 μM or 10 μM cold IP₃. Then, a specific binding value ofeach protein was obtained by subtracting the second radioactivity(non-specific binding value) from the first radioactivity values.

Scatchard plot analysis was conducted under the following conditions.For low-affinity polypetides (G224-m30 and control GST), the bindingexperiment was conducted in 100 μl binding buffer a by adding 9.6 nM[³H]IP₃ (DuPont NEN) and 10–20 nM of cold IP₃ to 2 μg of IP₃-bindingpolypeptide, and by adding 9.6 nM [³H]IP₃ (DuPont NEN) and 50 nM-2 μM ofcold IP₃ to 0.01 μg IP₃-binding polypeptide. For high-affinity IP₃sponges (G224 and G224-m49), binding experiment was conducted with 0.02μg IP₃ sponges at [³H]-IP₃ concentrations of 0.15, 0.3, 0.6, 1.2, 2.4,4.8 and 9.6 nM without adding cold IP₃.

The inhibition effects of the IP₃-binding polypeptides (IP₃ sponges) on[H]IP₃-binding activity of cerebellar microsome was analyzed as follows.

A microsomal fraction was prepared from the cerebella of mice ddY(Nippon SLC) mainly by following the method of Nakada et al. (Nakada S.et al., Biochem. J. 277:125–131, 1991). In 100 μl of binding buffer α,various concentrations of the IP₃ sponges were added respectively to seethe changes in the binding between the cerebellar microsome (40 μg) and9.6 nM [³H]-IP₃ according to the above method (see Scatchard plotanalysis).

As a result, the affinity of polypeptide G224 was found out to be 500times higher than that of polypeptide T734 (Kd=83 pM, B_(max)=1.6pmol/μg protein) (FIG. 3A). Polypeptide G224 binds well to (1,4,5)IP₃and (2,4,5)IP₃ and the yield of IP₃-binding protein was about 30 mg/lE.coli culture (Table 1). After purifying the protein with a glutathionecolumn and a subsequent PD10 column, the yield was about 24 mg/l. Thebinding activity was augmented when R441Q mutation was introduced intopolypeptide T734 (Yoshikawa et al., 1996, supra). The affinity ofpolypeptide G224-m49 (G224 introduced with R441Q mutation) doubled andbecame about 1,000 times higher than that of polypeptide T734 (Kd=about43 pM, B_(max)=1.7 pmol/μg protein) (FIG. 3B, Table 1). The bindingactivity decreased when polypeptide T734 was introduced with K508Amutation (Yoshikawa et al., 1996 (supra)). Similarly, the bindingactivity of polypeptide G224-m30 decreased when G224 was introduced withK508A mutation and became as low as about 1/4,000 of polypeptide G224and about 1/7,700 of polypeptide G224-m49 (Kd=about 330 nM, B_(max)=3.0pmol/μg protein) (FIG. 3C, Table 1).

(ii) IP₃-binding Inhibition via Absorption by Novel IP₃ Sponge

IP₃-binding polypeptides G224 and G224-m49 have powerful IP₃-bindingactivities that are 500 to 1,000 times higher than that of the originalIP₃ receptor. Polypeptides G224 and G224-m49 were tested for their useas an IP₃-specific absorption body (sponge) (IP₃ sponge), i.e., whetherthey can decrease the amount of IP₃-binding by the IP₃ receptors in asolution by competitively absorbing IP₃ in the solution (FIG. 4).

Mouse cerebellum is a tissue that is rich in IP₃ receptor and whosemicrosomal fraction has a [³H]IP₃-binding activity which is at least 50times higher than those in other tissues (Maeda et al., 1990 (supra)).Binding between 40 μg cerebellar microsome (Kd=21 nM, B_(max)=23 pmol/mgprotein) and 9.6 nM [³H]IP₃ in 100 μl solution was analyzed forpercentage (%) of competitive inhibition at various concentrations ofIP₃ sponges where the activity under the absence of IP₃ sponge wasconsidered 100%. It was calculated that, there were about 0.92 pmol ofIP₃-binding site of cerebellum IP₃ receptor and 0.96 pmol of [³H]IP₃present in the 100 μl solution.

As a result, no inhibition effect was observed for control GST even whenthe IP₃ sponge concentration was 100 μg/ml (FIG. 4). On the other hand,for high-affinity polypeptides G224 and G224-m49, strong IP₃-bindinginhibition activities were observed and IC₅₀ was about 10 μg/ml (FIG.4). Polypeptide G224-m30 with low affinity had low inhibition activitywith IC₅₀ of 100 μg/ml. According to this in vitro experiment system,the IP₃ sponge tended to precipitate with the microsome membrane whenthe IP₃ sponge concentration exceeded about 25 μg/ml, and so theconcentration-dependent curves were likely to fluctuate (FIG. 4). Thus,the apparent inhibition of G224-m30 observed at IP₃ sponge concentrationexceeding 25 μg/ml could be due to precipitation under highconcentration.

These results show that [³H]IP₃-binding of the IP₃ receptor canefficiently be inhibited according to the binding affinity and theconcentration of the IP₃ sponge used. High affinity IP₃-bindingpolypeptide of the invention is a novel IP₃ sponge that can be used asan IP₃ neutralizing agent, or an antagonist for IP₃-induced calcium.

Example 4 Test of Inhibiting IP₃-Induced Ca²⁺ Release (IICR)

To conduct a test of inhibiting IP₃-induced Ca²⁺ release, a microsomalfraction was prepared from mouse cerebellum as described in Example 3.The fraction was suspended in Buffer B, dispensed, and stored at −80° C.until it was used.

Composition of Buffer B was 110 mM KCl, 10 mM NaCl, 5 mM KH₂PO₄, 1 mMDDT, and 50 mM HEPES-KOH (pH 7.2) (containing a cocktail of proteaseinhibitors [0.1 mM PMSF, 10 μM leupeptin, 10 μM pepstatin A, 10 μM E-64]and 2 mM MgCl₂).

An IP₃-induced Ca²⁺ release activity of cerebellar microsome wasdetermined by using fura-2 (Molecular Probe) as a fluorescent Ca²⁺indicator. Specifically, excitations upon addition of IP₃ at twowavelengths (340 nm and 380 nm) were measured with fluorescencespectrophotometer CAF110 (Nihon Bunko) to see the change in thefluorescent intensity ratio (F340/F380) at 500 nm.

IP₃-induced Ca²⁺ release from the cerebellar microsome is generallyEC₅₀=100–200 nM IP₃. Cerebellar microsome (100 μg) was mixed with 500 μlof a release buffer (Buffer B containing 1 mM MgCl₂, 2 μM fura-2, 1 mMDTT, 10 mM creatine phosphate, 40 U/ml creatine kinase, 1 μg/mloligomycin, and the cocktail of protease inhibitors) in a measurementcuvette with a stirrer bar. The following reaction was conducted at 30°C. while constantly stirring with the stirrer bar.

One mM of ATP was added to the mixture in the cuvette to activate Ca²⁺pumping (Ca²⁺-ATPase), whereby Ca²⁺ was incorporated into the innerspace of microsome (Ca²⁺ loading). Ca²⁺ loading was confirmed bymonitoring until the decrease of fura-2 fluorescent level becameconstant. The change in the fura-2 fluorescent intensity ratio wasmeasured (F340/F380) at a subthreshold level.

The effect of IP₃ sponge on inhibiting IP₃-induced Ca²⁺ release activityof cerebellar microsome was analyzed as follows. After the addition ofATP, the curve of fura-2 fluorescent intensity was monitored until thedecrease became constant. Then, various concentrations of IP₃ spongeswere added. After 1 min., 50 nM to 1 μM of IP₃ was added to the reactionmixture to observe the change of fura-2 fluorescent intensity induced bythe IP₃.

The IP₃ sponge concentration dependency was determined as follows. Highaffinity polypeptide G224 of 3.125, 6.25, 12.5, 25, 50, 100, 200 μg/mlwere added to the reaction mixture, respectively. After about 1 min.,100 nM of IP₃ was added to measure the Ca²⁺ release activity induced bythe IP₃. The concentration dependency of low affinity polypeptideG224-m30 was determined by adding G224-m30 of 200, 400 and 500 μg/ml.After about 1 min., 100 nM of IP₃ was added to measure the Ca²⁺ releaseactivity induced by the IP₃. In addition, G224-m30 of 500 μg/ml was alsoadded, and after about 1 min., 50 nM of IP₃ was added to measure theCa²⁺ release activity induced by the IP₃.

As a result, it was found that the IP₃ sponges specifically inhibited ina competitive manner the IP₃-binding by the IP₃ receptor of cerebellarmicrosome by absorbing the IP₃ (FIGS. 5A–5F, 6A-6G and 7). In FIGS.5A–5F and 6A–G, the vertical axis represent the change in fura-2fluorescent intensity ratio (F340/F380) (i.e., change in the amount ofCa²⁺), and the horizontal axis represents the time (sec).

As shown in FIGS. 5A, 5B and 5C, in the absence of IP₃ sponge(controls), IP₃-induced Ca²⁺ release activities were dependent on IP₃concentration. Low-affinity polypeptide G224-m30 at a concentration of500 μg/ml had no inhibiting effect on Ca²⁺ release with 100 nM IP₃ (FIG.5E). Little difference was found between G224-m30 and the control foreffects on inhibiting 50 nM IP₃ (FIG. 5E). With GST only, even at a highconcentration of 632 μg/ml, no change was seen in Ca²⁺ release activityinduced with 50 nM IP₃ (FIG. 5F). Thus, in each case, no markeddifference was noted with the control.

On the contrary to the above results, high affinity polypeptide G224 hada significant inhibition effect on IP₃-induced Ca²⁺ release depending onits concentration (FIGS. 6A–6G). The high affinity polypeptide G224 hadthe greatest inhibition effect at 100 μg/ml and almost completelyinhibited the IP₃-induced Ca²⁺ release (FIG. 6F).

The peak values of Ca²⁺ release obtained by adding G224 at eachconcentration shown in FIGS. 6A–6G, were plotted where the peak obtainedin the absence of G224 was considered 100% (FIG. 7). The horizontal axisrepresents each concentration of polypeptide G224 and the verticle axisrepresents the peak value of Ca²⁺ release. As can be appreciated fromFIG. 7, the concentration of polypeptide G224 required for 50%inhibition of IP₃-induced Ca²⁺ release was about 20 μg/ml.

Accordingly, it was found that the high affinity IP₃-binding polypeptideacted as an IP₃ sponge and specifically inhibited, in aconcentration-dependant manner, the IP₃-induced Ca²⁺ release by the IP₃receptor on cerebellar microsome.

The present invention provides a polypeptide having a high affinitybinding activity to inositol 1,4,5-trisphosphate, a gene encoding thepolypeptide, a recombinant vector including the gene, a transformantincluding the vector and a method for producing the polypeptide.

The polypeptide of the invention can be used to control the inhibitionof a specific cell function that depends on an IP₃-induced calciumsignal transmission (IP₃ neutralizing agent, antagonist for IP₃-inducedcalcium, etc.). Furthermore, the polypeptide and the gene of the presentinvention is useful as an IP₃ signal detecting agent for inhibitingactivation of IP₃-induced calcium signal trasmission. The gene of theinvention is also useful as a therapeutic agent for treating a diseaseassociated with calcium production.

Various other modifications will be apparent to and can be readily madeby those skilled in the art without departing from the scope and spiritof the claims appended hereto be limited to the description as set forthherein, but rather that the claims be broadly construed.

All publications, patents, and patent applications cited herein areincorperated herein by reference in their entirety.

The following are information on sequences described herein:

What is claimed is:
 1. An isolated polypeptide consisting of the amino acid sequence of position 224 to 604 in SEQ ID NO:
 4. 2. An isolated polypeptide consisting of the amino acid sequence of position 224 to 604 in SEQ ID NO: 4, wherein the arginine at position 441 of SEQ ID NO: 4 is replaced with glutamine.
 3. An isolated polypeptide consisting of the amino acid sequence of position 1 to 604 in SEQ ID NO:
 4. 4. An isolated polypeptide consisting of the amino acid sequence starting at position 224 of SEQ ID NO: 4 and extending to at least position 579 of SEQ ID NO: 4 and at most position 604 of SEQ ID NO:
 4. 5. An isolated polypeptide consisting of a glutathione S-transferase and the amino acid sequence of position 224 to 604 in SEQ ID NO:
 4. 6. An isolated polypeptide consisting of a glutathione S-transferase and the amino acid sequence of position 224 to 604 in SEQ ID NO: 4, wherein the arginine at position 441 of SEQ ID NO: 4 is replaced with glutamine.
 7. An isolated polypeptide consisting of a glutathione S-transferase and the amino acid sequence starting at position 224 of SEQ lID NO: 4 and extending to at least position 579 of SEQ ID NO: 4 and at most position 604 of SEQ lID NO:
 4. 8. An inhibitor of an IP3-induced calcium release consisting of the polypeptide of any one of claims 1 and 2–7.
 9. A composition comprising the polypeptide of any one of claims 1 and 2–7 and a pharmaceutically acceptable carrier or additive.
 10. An IP3 binding protein as represented by the polypeptide of any one of claims 1 and 2–7.
 11. A method for inhibiting IP3-induced calcium release in a cell expressing an IP3 receptor protein comprising SEQ ID NO:4, the method comprising contacting the cell with the polypeptide of any one of claims 1 and 2–7. 